In our copending application Ser. No. 07/128,152, filed Dec. 3, 1987, and entitled "Cryoprotective Reagent", we have noted that carbohydrates have demonstrated the capacity to stabilize membrane structures under the severe conditions of dehydrations and freezing. These membranes are composed of ampiphilic lipid molecules in a bilayer arrangement. When the lipids associate with each other in such a configuration, they form a barrier with polar regions oriented toward the aqueous layers and a hydrophobic interior. This barrier constitutes the main structural arrangement of cell membranes and liposomal structures, which are often employed as model membrane systems or drug delivery systems.
In the absence of carbohydrates, membranes which are subjected to freeze drying, undergo fusion and intermixing of membrane lipids. In addition, the membranes lose their natural barrier properties and leak materials entrapped in the inner aqueous layer to the surrounding media. The use of carbohydrates added to the external aqueous media as well as the inner hydrophilic region, prevents these phenomena from occurring. Thus, membranes of cells or liposomes treated with carbohydrates maintain structural stability.
The amounts of carbohydrates employed to provide protection is on an equimolar level with the amount of membrane phospholipid present in the membranes. This imposes several limitations. Addition of the carbohydrate to the external media alone does not afford adequate protection. The levels of carbohydrate employed makes sample handling difficult and inefficient and impose potentially deleterious osmotic effects on the membrane.
Copending application Ser. No. 07/128,152 discloses and claims improved cryoprotectants comprised of a lipophilic molecule (such as a sterol), a molecule having polyhydroxyl groups (such as a carbohydrate), and a hydrophilic unit (such as polyoxyethylene) linking the lipophilic molecule with the molecule having polyhydroxy groups. These compositions elicit significantly more pronounced effects than free carbohydrates due to the attachment of the carbohydrate to the membrane directly. This alleviates problems associated with osmotic stress on the membrane and the difficulties encountered with the use of large amounts of material. The use of these agents also alleviates the problems associated with the placement of the carbohydrate on both the inner and outer faces of the membrane.
Crowe et al in "Factors affecting the stability of dry liposomes" [sic], Biochimica et Biophysica Acta 939 (1988), pp. 327-334, reported that a small amount of negatively charged lipid in the bilayer significantly increases stability. However, the negative charge in such compositions is membrane-bound and has adverse effects on circulation lifetimes of the liposomes.